Throughout this application various publications are referenced by full citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Pharmacological studies, and more recently gene cloning, have established that multiple receptor subtypes exist for most, if not all, neurotransmitters. The existence of multiple receptor subtypes provides one mechanism by which a single neurotransmitter can elicit distinct cellular responses. The variation in cellular response can be achieved by the association of individual receptor subtypes with different G proteins and different signalling systems. Further flexibility is provided by the ability of distinct receptors for the same ligand to activate or inhibit the same second messenger system.
Individual receptor subtypes reveal characteristic differences in their abilities to bind a number of ligands, but the structural basis for the distinct ligand-binding properties is not known. Physiologists and pharmacologists have attempted to specify particular biological functions or anatomical locations for some receptor subtypes, but this has met with limited success. Similarly, the biochemical mechanisms by which these receptors transduce signals across the cell surface have been difficult to ascertain without having well-defined cell populations which express exclusively one receptor subtype.
Receptors for epinephrine (adrenaline) are termed adrenergic receptors. The .alpha..sub.2 -adrenergic receptor belongs to the family of rhodopsin-like signal transducers which are distinguished by their seven-transmembrane configuration and their functional linkage to G-proteins. While all the receptors of the adrenergic type are recognized by epinephrine, they are pharmacologically distinct and are encoded by separate genes. These receptors, known as subtypes, are generally coupled to different second messenger pathways that are linked through guanine-nucleotide regulatory (G) proteins. Among the adrenergic receptors, .beta..sub.1 and .beta..sub.2 receptors activate adenylate cyclase, .alpha..sub.2 receptors inhibit adenylate cyclase and .alpha..sub.1 receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides (Chung, F. -Z., et al., J. Biol. Chem. 263:4052 (1988); Strader, C. D., et al., Proc. et al. Acad. Sci. USA 84:4384 (1987)).
Radioligand filtration binding techniques have been employed to characterize the adrenergic receptor family (Timmermans, P.B.M.W.M., ".alpha. Adrenoceptors", in Receptor Pharmacolocy and Function, Williams, M., Glennon R., and Timmermans, P. (eds.) 1989; Dekker, N.Y. pp. 173-205; Byland, D. B. TIPS 9:356 (1988)). Using these methods, two major classes of .alpha.-adrenoceptors have been described, .alpha..sub.1 and .alpha..sub.2. These differ in their selectivity for drugs. .alpha..sub.1 receptors can be labeled selectively with .sup.3 H-WB4101 or .sup.3 H-Prazosin. .alpha..sub.2 receptors can be labeled selectively with .sup.3 H-Yohimbine and .sup.3 H-Rauwolscine. Within the .alpha..sub.2 population, at least 3 subtypes have been defined, again on the basis of drug selectivity. All display high affinity for .sup.3 H-Yohimbine or .sup.3 H-Rauwolscine but differ in their susceptibility to competition by drugs. The .alpha..sub.2A subtype is very sensitive (nM) to competition by oxymetazoline. The .alpha..sub.2B subtype is sensitive to competition by Prazosin. The .alpha..sub.2C subtype is pharmacologically similar to the .alpha..sub.2B but .alpha..sub.2C has a higher (10 fold) affinity for .sup.3 H-Rauwolscine relative to that of the .alpha..sub.2B subtype.
Applicants have cloned a human, .alpha..sub.2B -adrenergic receptor, NGC-.alpha..sub.2B, which has been transfected into a heterologous expression system, producing a membrane protein with binding properties consistent with its preliminary characterization as an .alpha..sub.2 receptor subtype. The results from binding studies are consistent with the projected subtype based on amino acid sequence homology.
A variety of structural features which are invariant in the family of neurotransmitter molecules were present in clone NGC-.alpha..sub.2B. The greatest homology was found between clone NGC-.alpha..sub.2B and the human platelet .alpha..sub.2 and the human kidney .alpha..sub.2 -adrenergic receptors. (B. K. Kobilka, et al., Science 238:650-656, 1987; J. W. Regan, et al., Proc. Natl. Acad. Sci. (USA) 85:6301-6305, 1988). In both cases, an overall homology of approximately 45% was observed, while the homology within the transmembrane regions alone was approximately 75%.
The receptor encoded by clone NGC-.alpha..sub.2B shares numerous sequence and structural properties with the family of receptor molecules that has been predicted to span the lipid bilayer seven times. This family includes rhodopsin and related opsins (Nathans, J. and Hogness, D. S., Cell 34:807 (1983)), the .alpha. and .beta. adrenergic receptors (Dohlman, H. G., et al., Biochemistry 26:2657 (1987)), the muscarinic cholinergic receptors (Bonner, T. I., et al., Science 237:527 (1987)), the substance K neuropeptide receptor, (Masu, Y., et al., Nature 329:836 (1987)), the yeast mating factor receptors, (Burkholder, A. C. and Hartwell, L. H., Nucl. Acids Res. 13:8463(1985); Hagan, D. C., et al., Proc. Natl. Acad. Sci. USA 83:1418 (1986)); Nakayama, N. et al., EMBO J. 4:2643 (1985)), the serotonin receptor, and the oncogene c-mas, (Young, et al., Cell 45:711 (1986)). Each of these receptors is thought to transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Dohlman, H. G., et al., Biochemistry 26:2657 (1987); Dohlman, H. G., et al., Biochemistry 27:1813 (1988); O'Dowd, B. F., et al., Ann.Rev. Neuroscil, in press).